PROJECT SUMMARY Metals have complex effects on human health. Some are required in small amounts for normal development and homeostasis, and deficiencies can result in disease. However, overexposure to many metals poses even greater risks. Poisoning with essential and non-essential metals can lead to permanent neurological diseases, increased probability of degenerative syndromes, and acute organ injury. The bulk of metal poisoning in humans results from the consumption of food and water contaminated from inappropriate disposal of industrial wastes and in- adequate drinking water sanitation. Metal poisoning is also often an occupational hazard for industrial workers and miners, but most frequently, small children and poor communities suffer the highest incidence and most prolonged consequences of metal poisoning. Notably, genetic variation influences susceptibility to metal poison- ing, but the genomic factors that contribute to variation in resistance to metal poisoning represent a critically understudied area. Because resistance to metal poisoning is likely a genetically complex trait, substantial insight can be gained through genomewide dissection of quantitative natural variation. Our primary objective is to dissect and characterize the genetic variation underlying resistance to copper poisoning using the Drosophila melano- gaster model system, which shares several conserved metal-responsive genes and pathways with humans. We treat copper as a model metal of interest due to its critical requirement for normal cell function and the similarity of the copper metabolic pathway to that of both essential (such as zinc) and non-essential (such as lead) metals. With Aim 1 we will integrate a large-scale phenotyping screen for variation in copper resistance with tissue- specific expression profiling to identify and characterize loci and regulatory variants that contribute to copper resistance. With Aim 2 we will examine variation in the genetic architecture of copper resistance among 10 naturally segregating D. melanogaster populations, and understand how the genetic backgrounds and evolu- tionary history of these populations influences the effects of copper-associated allelic variation. With Aim 3, we will use genome editing and Reciprocal Hemizygosity Analysis to functionally validate strong candidate genes identified in Aims 1 and 2, directly testing the influence of specific alleles hypothesized to lead to high or low copper resistance in controlled genetic backgrounds. This integrated approach leverages (1) QTL mapping, gene expression analysis, and expression QTL mapping of copper resistance in a powerful reference mapping popu- lation, (2) bulked-segregant analysis of copper resistance in replicated naturally segregating populations, and (3) validation and characterization of candidate copper resistance genes through targeted gene editing. To- gether, these approaches will provide detailed insight into variation in the genetic control of resistance to metal poisoning. This will ultimately improve our understanding of susceptibility to metal poisoning in human popula- tions and foster innovation in the treatment of those who have been harmed by metal poisoning due to environ- mental exposure or metabolic disease.